A method for characterizing dissolved DNA and its application to the North Pacific Subtropical Gyre

Dissolved DNA (D-DNA) is a ubiquitous component of dissolved organic matter in aquatic systems. It is operationally defined as the DNA that passes a membrane filter and thus includes pools of truly dissolved "free" DNA (F-DNA), virion encapsidated DNA, DNA within membrane vesicles, and possibly other bound forms, each with different sources and lability. We investigated whether filtration (< 0.1 mu m), concentration by tangential flow ultrafiltration (> 30 kDa), and fractionation in an equilibrium buoyant density gradient could be used to discriminate the mass contributions of the different pools of filterable DNA in seawater. Spike-in experiments with a known range of DNA standards (75-20,000 bp) indicated that this method results in high recoveries of F-DNA (68-86%) with minimal degradation. Application of the fractionation method to seawater samples collected from the oligotrophic North Pacific Ocean followed by analysis of fractions (epifluorescence and electron microscopy, DNase digestion) suggested that the low-density fractions (1.30-1.35 g mL(-1)) were dominated by vesicle-like particles, mid-density fractions (1.45-1.55 g mL(-1)) by virus-like particles, and high-density fractions (1.60-1.70 g mL(-1)) by F-DNA. The estimated concentration of DNA that is either F-DNA, in viruses, or in vesicles was 0.13, 0.14, and 0.08 mu g L-1, respectively in the euphotic zone and 0.09, 0.04, and 0.03 mu g L-1, respectively in the mesopelagic zone. The approach described should be useful for more detailed investigations of the abundance, dynamics, and sources of DNA in the distinct pools that comprise filterable DNA in aquatic environments.